Tagged Questions

Questions pertaining to western blots, which are used for detecting specific proteins (sometimes called protein immunoblots).

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2
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1answer
33 views

How to prepare sample for SDS PAGE?

I usually take my protein sample 0.8ml and sample buffer(2X) 0.2ml for my sample preparation in SDS PAGE. Am I using correct proportion? my protein sample concentration is 4.1mg/ml. What is the ...
3
votes
1answer
39 views

What equation to compare protein isoforms in a Western Blot?

The protein isoforms I am interested in comparing appear as distinct bands on the gel I have already run. I have an Excel sheet with optical density measurements I obtained using ImageJ; it looks ...
1
vote
1answer
39 views

Can I use grayscale images when working with ImageJ?

I am using ImageJ to analyze Western Blots. I have scanned films in as grayscale images because this is how we did it in my old lab. People in my current lab are not satisfied with that explanation ...
1
vote
1answer
31 views

can a bacterial protease inhibitor cocktail be used for Western Blotting involving HUVEC cells and HT-29 adenocarcinoma cells?

Dear fellow biochemists, I need some advice on Western Blotting, more specifically the use of certain protease inhibitors with the RIPA cell lysis buffer and protease inhibitor cocktail. A Millipore ...
2
votes
2answers
41 views

Western Blot: Fischer ECL substrate 2:1 instead of 1:1?

i recently developed a b-actin-ab-incubated PVDF-WB-Membrane with the Thermo Scientific Pierce ECL Kit. According to the manufacterers orders i mixed Reagent 1 and Reagent 2 1:1. But unfortunately i ...
3
votes
1answer
90 views

How to remove bad lanes in ImageJ Westernblot analysis

I use ImageJ to do an analysis of a Westernblot Image. If everything goes as wanted the workflow is fine. But if I do something wrong creating a lane there is no undo for a lane and also no way to ...
1
vote
0answers
41 views

Meta-Data Retrieval from Bio-Rad Images

I asked a related question on SO, but I also had some biology related questions to this issue. One of the big things I'd like to do is sort between DNA/UV images and Western/Chemi images. After ...
3
votes
1answer
32 views

Prevalent large (>=90kDa) maintenance protein/loading control

I was wondering if anyone had recommendations for good, large (hopefully 100kDa+), control proteins that would be present in most mammalian cells. I'm working mostly with tissue samples from humans ...
4
votes
1answer
167 views

Western blotting questions?

everyone. I've just been introduced to the procedure of Western blotting from my reading, though I'm not entirely sure about some points. I'd appreciate it if someone could help me with this. What ...
1
vote
1answer
52 views

Enzymatic Protein Deglycosylation of TCL

I've done O- and N- linked enzymatic deglycosylation of purified and lyophilized proteins. Following some modifications to the manufacture's protocol, I had great results. I now want a student to ...
5
votes
2answers
309 views

Can this image be the result of a Western Blot?

I am currently researching image manipulation in the life sciences and stumbled upon a Western Blot looking very similar to the image below. For creating the image I have cut out the blot using the ...
5
votes
2answers
348 views

High Current (Speed) Transfer Buffer Recipe

Does anyone know an effective buffer mix to use for high current Western transfers? We are successfully using the vendor's premixed buffer to transfer a wide range of protein sizes to PVDF membranes ...
0
votes
0answers
53 views

Western blotting of lung samples [duplicate]

I have been working on lung samples to perform western blotting. I have two related questions about this: I get very nonspecific results: at times nothing at all, at times it glows on membrane but ...
5
votes
2answers
108 views

Is there a strong reason to be sceptical about the “cured HIV patient” being reported by mainstream media?

There's a story going round the news about a baby that was, apparently, cured of HIV using a cocktail of drugs at an early age. The story piqued my interest, but details seem scarce. One of the main ...
5
votes
1answer
326 views

How to do western analysis to lung tissue?

I am working with lungs from mice, and I want to do a western blot analysis to my samples. I am having trouble with this because my images turn out very "messy", with a high background. Perhaps I ...